Efficient Method of Agrobacterium-mediated Transformation for Triticale (x Triticosecale Wittmack)
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Transgenic plants of triticale cv. Wanad were obtained after transformation using three combinations of strain/vectors. Two of them were hypervirulent Agrobacterium tumefaciens strains (AGL1 and EHA101) with vectors containing bar under maize ubiquitin 1 promoter (pDM805), and both hpt under p35S and nptII under pnos (pGAH). The third one was a regular LBA4404 strain containing super-binary plasmid pTOK233 with selection genes the same as in pGAH. The efficiency of transformation was from 0 to 16% and it was dependent on the selection factor, auxin pretreatment, and the strain/vector combination. The highest number of transgenic plants was obtained after transformation with LBA4404(pTOK233) and kanamycin selection. Pretreatment of explants with picloram led to the highest number of plants obtained after transformation with both Agrobacterium/vector systems LBA4404(pTOK233) and EHA101(pGAH) and selected with kanamycin. Transgenic character of selected plants was examined by PCR using specific primers for bar, gus, nptII, and hpt and confirmed by Southern blot hybridization analysis. There was no GUS expression in T0 transgenic plants transformed with gus under p35S. However the GUS expression was detectable in the progeny of some lines. Only 30% of 46 transgenic lines showed Mendelian segregation of GUS expressing to GUS not expressing plants. In the remaining 70% the segregation was non-Mendelian and the rate was much lower than 3:1. Factors that might effect expression of transgenes in allohexaploid monocot species are discussed.
KeywordsTriticale Agrobacterium tumefaciens Cereal transformation Transgene expression PCR analysis
We thank Dr. Yukoh Hiei from Japan Tobacco Inc for supplying pTOK233 plasmid, Prof. Norio Murata (National Institute for Basic Biology, Japan) for supplying pGAH vector, and Dr. Richard Brettell (CSIRO Plant Industry) for sending pDM805 plasmid. The research was supported by the 5FP of European Community contract No QLK3-CT-2000-00078 and Polish Committee of Scientific Research grant 5 P06A 02117.
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